Nitrogen deprivation causes diploid cells of the yeast S. cerevisiae to depart from vegetative growth and form pseudohyphae. These are filaments of rod-shaped cells that radiate from the periphery of a colony and spread over and into the agar medium. The transition arises when a yeast form (YF) cell divides and gives rise to a PH daughter that has a different developmental fate from its mother. This asymmetric switch is fundamentally the same as those that occur during the production of distinct lineages from pluripotent stem cells in humans. In yeast as in mammals, the fate change requires co-ordination of programs that are controlled by multiple transcription factors. Some are responsive to extracellular stimuli and others on cell inherent mechanisms for asymmetric segregation of the critical fate determining factors. S. cerevisiae Ash 1 is a GATA-family transcription factor that is differentially segregated to daughters and is essential for the PH transition. While the mechanism responsible for asymmetric localization of Ash1 has been elucidated, very little is known about the daughter-specific program that it mediates to establish the PH fate. This program has been difficult to study by standard methods because PH colonies are a heterogeneous mixture of cell-types. The applicant proposes to develop strategies using green fluorescent protein (GFP) labels that allow analyses of promoter function and protein and mRNA dynamics in individual living cells. Currently, the cell-surface adhesion protein, Flo11, is the only known gene product required for PH growth whose transcription is dependent on Ash1. Aim (1) focuses on Flo11 transcriptional regulation. Studies will define the Ash 1-dependent mechanism at this promoter and reveal how Ash1 collaborates with signal dependent transcriptional regulators of Flo11. Aim (2) addresses daughter-specific localization and functions of Flo11 in PH growth. Aim (3) proposes a novel strategy to generate daughter-specific complementary deoxyribonucleic acids (cDNAs) for use in array analyses to identify daughter-specific genes subject to Ash1 control. The principles the applicants establish for the mechanism by which Ash1 transcriptional control is integrated with that of signal dependent transcription factors will be broadly applicable to the GATA-family. In particular, Ash1 is conserved and required for the YF-PH transition that is essential for virulence of pathogenic fungi. Thus, the molecular mechanisms Dr. Errede defines will be directly applicable to those underlying fungal pathogenicity. Additionally, the project has the potential to reveal fungal proteins that would be excellent candidates as targets for drug intervention.